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Abstract:

A device for performing chromatographic separations and nuclear magnetic
resonance analysis has trapping means for holding a separated sample and
to form a held separated sample and placing said held separated sample in
said nuclear magnetic resonance assembly. One preferred trapping means
forms a held separated sample and a passed separated sample. The passed
separated sample is discharged from the device. Preferred trapping means
comprise a trapping column or a separated sample loop.

Claims:

1. A device for performing a chromatographic separation and a nuclear
magnetic resonance analysis on a sample comprising: a. a closed
chromatographic assembly having an input, an outlet, and chromatographic
device said input for receiving one or more samples, said output for
discharging one or more separated samples and said chromatographic device
for separating said sample to form one or more separated samples having
retention time data; b. conduit means in fluid communication with said
closed chromatographic assembly for conveying said one or more separated
samples to a nuclear magnetic resonance assembly; c. a nuclear magnetic
resonance assembly for receiving one or more separated samples defined by
retention times and producing nuclear magnetic resonance data for said
one or more separation samples; d. control means in signal communication
with said closed chromatographic assembly and said nuclear magnetic
resonance assembly to receive retention time data and nuclear magnetic
resonance data and associating said retention time data and nuclear
magnetic resonance data to at least one of said sample and said separated
sample.

2. The device of claim 1 wherein said conduit means has trapping means
for holding a separated sample to form a held separated sample and
placing said held separated sample in said nuclear magnetic resonance
assembly.

3. The device of claim 2 wherein said trapping means forms a held
separated sample and a passed separated sample, said passed separated
sample discharged from said device.

4. The device of claim 3 wherein said trapping means is a trapping
column.

5. The device of claim 3 wherein said trapping means is a separated
sample loop.

6. The device of claim 2 wherein said trapping means is in fluid
communication with nuclear magnetic resonance reagents and said trapping
means releases said held separated sample in said nuclear magnetic
resonance reagents to form one or more deuterated separated samples for
nuclear magnetic resonance analysis.

7. The device of claim 1 further comprising a second detector in fluid
communication with said conduit means and in signal communication with
said control means, said second detector producing second detector data,
said control means associating said second detector data with said
retention time data and nuclear magnetic resonance data to at least one
of said sample and said separated sample.

8. The device of claim 7 wherein said second detector is a mass
spectrometer.

9. The device of claim 7 wherein said second detector is a photo diode
array detector.

10. The device of claim 7 further comprising a peak detector said peak
detector in fluid communication with said conduit means and in signal
communication with said control means, said peak detector producing one
or more signals corresponding with an analyte of interest or a potential
analyte of interest in a separated sample to isolate said separation
sample to form an isolated separated sample and direct said isolated
separated sample to at least one of said nuclear magnetic resonance and
said second detector.

11. The device of claim 10 further comprising valve means in fluid
communication with said conduit means, said valve means for receiving
said isolated separation sample and forming isolated separated sample
aliquots and directing at least one isolated separated sample aliquot to
said nuclear magnetic resonance and said second detector such that said
isolated separated sample aliquot is associated by control means with
nuclear magnetic resonance data, said second detector data and retention
time data.

12. A method of performing a chromatographic separation and a nuclear
magnetic resonance analysis on a sample comprising the steps of: A.
providing a device having a closed chromatographic assembly, conduit
means, a nuclear magnetic resonance assembly and control means, i. said
closed chromatographic assembly having an input, an outlet, and
chromatographic device said input for receiving one or more samples, said
output for discharging one or more separated samples and said
chromatographic device for separating said sample to form one or more
separated samples having retention time data; ii. said conduit means in
fluid communication with said closed chromatographic assembly for
conveying said one or more separated samples to said nuclear magnetic
resonance assembly; iii. said nuclear magnetic resonance assembly for
receiving one or more separated samples defined by retention times and
producing nuclear magnetic resonance data for said one or more separation
samples; iv. said control means in signal communication with said closed
chromatographic assembly and said nuclear magnetic resonance assembly to
receive retention time data and nuclear magnetic resonance data and
associating said retention time data and nuclear magnetic resonance data
to at least one of said sample and said separated sample; B. operating
said device to produce nuclear magnetic resonance data and retention time
data associated with at least one of a sample and a separated sample.

13. The method of claim 12 wherein said conduit means has trapping means
for holding a separated sample to form a held separated sample and
placing said held separated sample in said nuclear magnetic resonance
assembly.

14. The method of claim 13 wherein said trapping means forms a held
separated sample and a passed separated sample, said passed separated
sample discharged from said device.

15. The method of claim 14 wherein said trapping means is a trapping
column.

16. The method of claim 14 wherein said trapping means is a separated
sample loop.

17. The method of claim 13 wherein said trapping means is in fluid
communication with nuclear magnetic resonance reagents and said trapping
means releases said held separated sample in said nuclear magnetic
resonance reagents to form one or more deuterated separated samples for
nuclear magnetic resonance analysis.

18. The method of claim 12 further comprising a second detector in fluid
communication with said conduit means and in signal communication with
said control means, said second detector producing second detector data,
said control means associating said second detector data with said
retention time data and nuclear magnetic resonance data to at least one
of said sample and said separated sample.

19. The method of claim 18 wherein said second detector is a mass
spectrometer.

20. The method of claim 18 wherein said second detector is a photo diode
array detector.

21. The method of claim 18 further comprising a peak detector said peak
detector in fluid communication with said conduit means and in signal
communication with said control means, said peak detector producing one
or more signals corresponding with an analyte of interest or a potential
analyte of interest in a separated sample to isolate said separation
sample to form an isolated separated sample and direct said isolated
separated sample to at least one of said nuclear magnetic resonance and
said second detector.

22. The method of claim 21 further comprising valve means in fluid
communication with said conduit means, said valve means for receiving
said isolated separation sample and forming isolated separated sample
aliquots and directing at least one isolated separated sample aliquot to
said nuclear magnetic resonance and said second detector such that said
isolated separated sample aliquot is associated by control means with
nuclear magnetic resonance data, said second detector data and retention
time data.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of International Application No.
PCT/US2010/031194, filed on Apr. 15, 2010. This application is also a
continuation-in-part of International Application No. PCT/US2010/030698
filed on Apr. 12, 2010, which claims benefit of a priority to U.S.
Provisional Application No. 61/168,306 filed on Apr. 10, 2009. The
contents of all these applications are incorporated herein by reference
in their entirety.

FIELD OF THE INVENTION

[0002] Embodiments of the present invention are directed to apparatus and
methods for performing liquid chromatography separations coupled to
nuclear magnetic resonance (NMR) analysis in which the sample separated
by liquid chromatography is received directly for NMR analysis.

BACKGROUND OF THE INVENTION

[0003] The increasingly widespread use of advanced spectroscopic detectors
such as mass spectrometers has dramatically broadened the utility and
information-yield of analytical liquid chromatographic separations. Older
"conventional" detection techniques such as refractometry or
fixed-wavelength ultraviolet-absorbance detection might imply the
presence of a suitable analyte within the detection volume, and with the
use of known calibrants, might imply the concentration of that analyte.
However, the identity of the analyte, at best, was inferred by comparison
with the chromatographic retention time or retention volume of a known
standard. Identification of the analyte was effectively not a property or
a capability of those older detection subsystems.

[0004] Such detectors were also susceptible to significant quantitation
errors in the presence of overlapping chromatographic bands or zones,
limiting their utility in the analysis of highly-complex mixtures as are
commonly encountered in biological and environmental applications.
Historically, the application of mass spectrometry to the task of liquid
chromatography detection facilitated analyte detection within vastly more
complex mixtures, by permitting high-resolution separation in the
mass-to-charge domain to augment chromatographic separation in the
liquid-volume domain.

[0005] Depending upon the mode of mass spectrometric analysis being
performed, and the nature of the analyte(s), putative compound
identification might be performed in-line during the separation,
substantially without reliance upon the chromatographic retention time or
retention volume of a standard. There exist, however, important classes
of compounds for which mass spectrometry, alone, is not capable of
rendering a full and complete identification. Commonly, examples are
found where isomerization is present, where the isomers share or exhibit
the same chemical formula and parent mass-to-charge-ratio, but are
assembled in arrangements which may be either structurally or spatially
distinct. Knowledge of the chemical formula, while useful, is incomplete
if the configuration or arrangement of the molecule is questionable or
fully unknown. Isomers may have distinctly (in some cases, radically)
different behaviors within biological systems, if supplied as
pharmaceutical compounds, or if rendered as degradation products. The
fates of important contaminant materials in the environment may entail
molecular rearrangements of multiple types.

[0006] Nuclear Magnetic Resonance ("NMR") spectroscopy is a powerful
complement to mass spectroscopy in the analytical toolkit. The structural
information yielded by NMR spectra can be used to infer how the molecule
is arranged. However, the requirements for NMR analysis have made it
difficult to directly couple NMR analysis with other detectors and
chromatographic techniques. In this context, the term "directly coupled"
means a single sample is separated by chromatographic means to form a
separated sample and one or more aliquots of the separated sample are
received by spectrometric and NMR analysis without an intervening
collection into containers such as vials, well-plates, and the like. The
use of vials, well-plates, or other fraction-collection devices implies
an "off-line" analytical methodology, in contrast to a directly-coupled
methodology. In a directly-coupled method, the analyte sample borne in
solution in a mobile phase is conveyed within fluid conduits throughout
that analytical method, and typically does not emerge into a receiving
vessel such as a vial until it has traversed the entirety of that method.
A vial may be used to collect waste from a first analytical process or
method, or a vial may be used to capture the sample for transport and
submission to a subsequent analytical method implemented as a separate
system. The subsequent system would be referred to as operating
"off-line" from the first system.

SUMMARY OF THE INVENTION

[0007] Embodiments of the present invention directly couple a liquid-phase
separation (liquid chromatography, or "LC") with what is effectively an
in-line structure-elucidation capability. This coupling will plausibly
form a fundamentally important component of the toolkit of analysts
working in pharmaceutical, environmental, homeland-security, natural
products, food/agriculture, or forensic application areas.

[0008] One embodiment of the present invention is directed to a device for
performing a chromatographic separation and a nuclear magnetic resonance
analysis on a sample. The device comprises a closed chromatographic
assembly having an input, an outlet, and a chromatographic device. The
input is for receiving one or more samples. The output is for discharging
one or more separated samples. The chromatographic device is for
separating the sample to form one or more separated samples having
retention time data. The device further comprises conduit means in fluid
communication with the closed chromatographic assembly for conveying the
one or more separated samples to a nuclear magnetic resonance assembly.
The device further comprises a nuclear magnetic resonance assembly for
receiving one or more separated samples defined by retention times and
producing nuclear magnetic resonance data for the one or more separated
samples. The device further comprises control means in signal
communication with the closed chromatographic assembly and the nuclear
magnetic resonance assembly to receive retention time data and nuclear
magnetic resonance data and associating the retention time data and
nuclear magnetic resonance data to at least one of the sample and said
separated sample.

[0009] The term "chromatographic assembly" is used to refer to equipment
and ancillary devices for performing chromatographic separations. As used
herein, the term "chromatographic device" refers to a column, cartridge,
capillary or other inline plumbed separation device. Such separation
devices are typically packed with particles, beads, porous monolith and
the like, although capillary type devices may rely on internal wall
structures. Thus, the chromatographic assembly performs separations under
pressure, in the manner of high pressure liquid chromatography, and even
higher pressure liquid chromatography, above 5,000 or 6,000 psi, gas
chromatography and super critical fluid chromatography.

[0010] As used herein, the term "control means" refers a computer or CPU
and supporting software, firmware and instructions. The computer or CPU
may be a personal computer, mainframe, server or integral with one or
more assemblies of the device. The term "signal" or "signal
communication" is used in an optical, electrical, magnetic or mechanical
sense to denote wired, radio or photo communication and signaling.

[0011] The term "nuclear magnetic resonance assembly" is used to refer to
equipment and ancillary devices for performing nuclear magnetic resonance
analyses.

[0012] The term "conduit means" refers to tubing, piping, conduits,
capillaries and all associated fittings, valves, and ancillary supporting
components and the like for placing components and assemblies in fluid
communication. As used herein, the term "fluid communication" means
plumbed together, as in linked by pipes, tubing and the like, to move
fluids there between.

[0013] One preferred conduit means has trapping means for holding a
separated sample to form a held separated sample and placing said held
separated sample in said nuclear magnetic resonance assembly. One
preferred trapping means forms a held separated sample and a passed
separated sample. The passed separated sample is discharged from the
device. Preferred trapping means comprise a trapping column or a
separated sample loop. A trapping column may be packed with a stationary
phase constructed and optimized so as to exhibit highly retentive
behavior for classes of compounds referred to above as "held separated
sample".

[0014] A preferred trapping means is in fluid communication with nuclear
magnetic resonance reagents. And, the trapping means releases the held
separated sample in the nuclear magnetic resonance reagents to form one
or more deuterated separated samples for nuclear magnetic resonance
analysis.

[0015] One preferred device further comprises a second detector in fluid
communication with the conduit means and in signal communication with the
control means. The second detector produces second detector data, and the
control means associates the second detector data with the retention time
data and nuclear magnetic resonance data to at least one of said sample
and said separated sample. A preferred second detector is a mass
spectrometer or a photodiode array optical-absorbance detector.

[0016] A preferred device further comprises a peak detector. The peak
detector is in fluid communication with the conduit means and is in
signal communication with the control means. The peak detector produces
one or more signals corresponding with an analyte of interest or a
potential analyte of interest in a separated sample to isolate the
separation sample to form an isolated separated sample. The peak detector
directs the isolated separated sample to at least one of the nuclear
magnetic resonance and the second detector. Preferably, the device
further comprising valve means in fluid communication with the conduit
means to facilitate the directing of the isolated separated sample. One
preferred valve means is for receiving the isolated separation sample and
forming isolated separated sample aliquots and directing at least one
isolated separated sample aliquots to the nuclear magnetic resonance and
the second detector such that said isolated separated sample aliquot is
associated by control means with nuclear magnetic resonance data, the
second detector data and retention time data. As used herein, valve means
refers to one or more valves used singularly, or in groups.

[0017] A further embodiment of the present invention is directed to a
method of performing a chromatographic separation and a nuclear magnetic
resonance analysis on a sample. The method comprises the steps of
providing a device as previously described, having a closed
chromatographic assembly, conduit means, nuclear magnetic resonance
assembly and control means; and, operating the device to produce nuclear
magnetic resonance data and retention time data associated with at least
one of a sample and a separated sample.

[0018] Embodiments of the present invention permit structure-elucidation
of small-molecules. For example, embodiments featuring a nuclear magnetic
resonance assembly having relatively simple 1D proton-NMR mode, and a
relatively simple spectrometer of modest size (at-bench magnetic
resonance detector or "MRD", with a substantially single-board
electronics implementation, and active- or passive-shielding for the
stray field of the magnet), if sufficient sample (and sample
concentration) is available, may be employed to carry out the NMR portion
of the analysis. In small-molecule analysis, it is the coupling of mass
spectrometry and NMR spectroscopic techniques which effectively underpins
and enables structure-elucidation. The rules of such elucidation, at
least for small molecules, are sufficiently well known that commercial
software packages have been written to accomplish this in a substantially
automated manner (see, for example,
http://www.acdlabs.com/products/spec_lab/complex_tasks/str_elucidator/
from Advanced Chemistry Development Inc., Toronto, Ontario, Canada M5C
1T4).

[0019] "Offline" NMR spectroscopy of properly-prepared samples resident in
vials ("NMR tubes") is an established technique within dedicated NMR
laboratories. The coupling of NMR spectroscopy with liquid chromatography
introduces a different set of problems than those encountered in the
coupling of mass spectrometry with liquid chromatography. Mass
spectrometric analysis typically has the sensitivity and speed to
interface directly in real-time with a liquid chromatography separation
(whether high-performance liquid chromatography ("HPLC"), or
ultra-performance liquid chromatography (such chromatography associated
with "HPLC®" equipment sold by Waters Corporation, Milford, Mass.,
USA). NMR spectrometers make use of inherently weaker signals, and
typically require both a meaningfully larger sample mass (and sample
concentration) to be present for analysis, and typically require a longer
sample interrogation time than may be accommodated by the chromatography.
Moreover, achievement of a usefully-large signal-to-noise ("S/N") ratio
in the proton NMR of dissolved species requires the substitution of a
deuterated solvating phase for the normally non-deuterated solvating
phase in which the sample is typically chromatographed (i.e. D2O is
substituted for H2O, CD3CN is substituted for CH3CN,
DMSO-d6 is substituted for DMSO (dimethyl sulfoxide), etc.). The latter
requirement emerges because the solvent species is present within the
detector cell at a concentration many orders-of-magnitude larger than the
concentration of the solvated analyte. The proton signal-of-interest is
that which is associated with the analyte, not that which is associated
with the solvent. Reduction of the solvent background signal is the
intent of deuterated-solvent substitution. At a separation scale
corresponding to that of capillary chromatography, it is feasible to run
the entire chromatography separation in deuterated phases, and we have
done so, but this practice is not feasible across-the-board in either a
typical analytical laboratory context, or (most certainly) within a
typical preparatory chromatography context.

[0020] Embodiments of the present invention feature conventional-scale LC
accomplished with columns of typically 2.1 mm internal diameter, and
chromatographic pressures which may be greater than 5,000 PSIG, or a
supercritical fluid chromatography ("SFC") separation technique, coupled
with an NMR spectroscopic technique. Embodiments of the present invention
feature a primary chromatographic separation of a peak-of-interest
corresponding to a potential analyte-of-interest, and the temporary
isolation and accumulation of that analyte-of-interest within a fluid
conduit offline from the separation stream, or accumulating from a single
or from a plurality of LC runs, the transfer of the analyte-of-interest
from that fluid conduit to a trapping column containing a stationary
phase on which the analyte can be retained and focused, purge-out of
protonated solvent from the trapping column, trapping valve, and
associated tubing by a first deuterated phase (typically D2O for the
case of a reversed-phase trapping mode), when deuteration of the fluid
environment of the trap is substantially complete, step-elution of the
analyte from the trapping column is accomplished by a second deuterated
phase (optionally deuterated DMSO (DMSO-d6) or deuterated acetonitrile
(CD3CN) in the case of a reversed-phase LC trapping mode), where the
analyte is thereby conveyed downstream to an NMR spectrometer flow-probe
as a highly-concentrated step-elution band, and "parking" of that focused
band within the interrogation region of the detector cell of the
flow-probe of the NMR spectrometer, until NMR spectroscopy is complete.
Upon completion of NMR spectroscopy, the analyte is "unparked" or
migrated out of the flow probe, typically to a waste receptacle, although
other routes of emergence from the flow probe may be readily contemplated
if the analyte is to be further used.

[0021] In the context of the above description, it is helpful to introduce
the concept of "impedance matching" between the LC separation and the
detector cell of the NMR probe. To achieve a suitably high degree of
sensitivity in the NMR analysis (i.e. to be able to carry out the NMR
analysis with a suitably small mass of analyte material) it is
advantageous to make use of a microcoil NMR probe configuration. Such
probes are commercially available through Magnetic Resonance Microsensors
(MRM) Corporation (Savoy, Ill., USA), a division of Protasis Corporation
(Marlborough, Mass., USA). Microcoil probes achieve high S/N with
relatively low sample mass requirements through the use of a very small
detection-cell volume (typically 2.5 or 5.0 microliter interrogated
volume) which is encompassed within a correspondingly-small
excitation-and-detection radiofrequency (RF) coil. To make appropriate
use of such a cell, the chromatography zone or band conveying the
dissolved analyte must be of few-microliter volume, and the analyte must
be present at sufficiently high concentration that an appropriate number
of analyte molecules is present within that few-microliter volume.
Typically, analyte concentrations at the milliMolar (mM) level (1 to 30
mM typically) are required, depending upon the nature of the NMR
spectroscopy being performed. One to thirty milliMolar analyte
concentrations are higher than what is typically manipulated within
analytical LC separations, and zones or bands of few-microliter volume
are smaller than what is typically manipulated in conventional analytical
LC separations. The concept of "impedance matching" in this context will
be defined below. Impedance matching is an electrical design practice
wherein properties of an electrical load and an electrical source are
matched so as to maximize the power transfer between the two, and
minimize reflections from the load. Impedance matching is a useful
concept which is discussed in contexts other than electrical design.
Examples include acoustic impedance matching, optical impedance matching,
and mechanical impedance matching. In the coupling of LC with microcoil
NMR spectroscopy, one is confronted with a situation where analyte in the
LC process resides at lower concentration and at larger volume than is
useful for insertion into a microcoil probe. The microcoil probe requires
relatively intense analyte concentrations to exist in relatively minute
volumes. In common between the two is the presence of a total analyte
mass, which one would like to convey from one process (LC) to a second
process (NMR) with high efficiency and with little-or-no loss or wastage.
We introduce here the concept of impedance matching between (for example)
a 2.1 mm internal diameter column LC separation, and a microcoil probe.
Such impedance matching makes use of chromatographic principles, and
incorporates at least one additional column device containing a retentive
phase, beyond the primary separation column. In usage sequences detailed
below, this additional column device is selected to be highly retentive
for the classes of analyte of interest, and to have a relatively small
bed volume. The purpose of this column device, which may be referred to
as a trap or trapping column, is to process a relatively dilute incoming
sample fractionated or sliced from a primary chromatography separation,
where such processing results in the analyte becoming immobilized (and
thus distributed over) a retentive bed of relatively small volume. Once
immobilized there, the analyte can be washed of salts or other
mobile-phase modifiers which might be present in the primary
chromatography separation, and which may disrupt or degrade the quality
of the NMR spectroscopy. Protonated solvent may also be washed away while
the analyte-of-interest is retained on the trap bed. Washing steps may
optimally make use of inert-gas purges between liquid-phase introduction
steps. When washing of the retained analyte is complete, a final
inert-gas purge step is undertaken, prior to analyte elution. In a
preferred embodiment, the trap is packed with the stationary phase
Oasis® HLB (Waters Corporation, Milford, Mass., USA), and the elution
solvent is DMSO-d6. Following inert-gas purging, the elution solvent
arrives in a sharp "front", and conveys the analyte downstream in a
densely-concentrated band. The analyte concentration and volume within
this new band are now appropriate for matching with the properties of the
microcoil probe. This low-volume, high-concentration eluted band is
conveyed to the microcoil NMR probe and parked there for the duration of
the NMR analysis. Absent this impedance-matching function, the analyte as
eluted from the primary chromatography separation would be poorly and
inadequately utilized by a microcoil NMR probe, and little overall
analytical utility would be achieved.

[0022] While a reversed-phase mode of analyte trapping is described above,
operating in conjunction with a reversed-phase mode of chromatographic
separation in the primary chromatography system, it is readily envisioned
that other trapping modes may be employed, such as normal-phase trapping,
or the use of specialized stationary phases and separation techniques as
appropriate to the separation of chiral compounds.

[0023] Sample trapping on a sorbent re-focuses the peak and permits
exchange for deuteration. With trapping, there is no requirement to
manually dry the sample in a vial and re-solvate it in a new (deuterated)
solvent system. The trapping asset and related system control enable full
system automation, where a run can be properly configured, and a
structure elucidated as a result (i.e. the analyst may configure an
analysis, and return to be presented with "the answer" (an elucidated
structure) from this automated system, not just a chromatogram or other
lower-level ensemble of data).

[0024] A preferred trapping sorbent is packed in a column housing which is
non-magnetic, and connected to the chromatography system using tubing
which is similarly non-magnetic, such that the trap can be positioned
very close to the flow-probe entry-point. Thus the distance intervening
between the NMR spectrometer and the primary chromatography system can be
bridged by a solvent flow where, by design, analyte is re-focused
proximal to the entrance to the NMR flow-probe. Substantial elimination
of the zone-broadening associated with analyte transport to the NMR
spectrometer is an enabling capability to ensure that analyte
concentration within the NMR detector cell is maximized.

[0025] From our experience, we recognize that elution of analyte on a
relatively sharp step-gradient "front" may present a
solvent-susceptibility mismatch when NMR probe shimming is underway. This
matter is discussed in some detail in U.S. Pat. No. 6,404,193, which is
of common inventorship with the present disclosure, and which is
incorporated herein by reference. It is anticipated that the fluid path
residing between the trap column and the outlet of the NMR detector cell
may, under automated system control, be pre-filled with the "strong"
(i.e. organic) deuterated solvent which is subsequently used to elute the
analyte from the trap. Only after this pre-filling is accomplished, is
the trap valve switched such that the deuterated organic flow can elute
the analyte from the trap. The pre-filling of the fluid path downstream
of the trap column is intended to substantially reduce the magnitude of
the solvent discontinuity which exists at or near the NMR detector cell
at the time of detection, thereby reducing or partially mitigating
spectral line-broadening associated with susceptibility mismatch.

[0026] These and other features and advantages of the present invention
will be apparent to those skilled in the art upon reading the detailed
description that follow and viewing the Figures.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 depicts in schematic form a device incorporating features of
the present invention;

[0028]FIG. 2 depicts in schematic form a device incorporating features of
the present invention;

[0029] FIG. 3 depicts in schematic form a device incorporating features of
the present invention; and,

[0030]FIG. 4 depicts in schematic form a device incorporating features of
the present invention.

[0031]FIG. 5A is a more-detailed view of valve components and fluidic
interconnections used to implement conduit means 15 as introduced in the
context of the system of FIG. 1.

[0032]FIG. 5B is a supplementary detailed view of valve components and
fluidic interconnections used to implement conduit means 15 as introduced
in the context of the system of FIG. 1.

[0033]FIG. 6A is an overview of detector data streams available to the
system controller for diagnostic or other purposes.

[0034]FIG. 6B is an expanded view of minutes 4 to 9 of the detector data
streams of FIG. 6A.

DETAILED DESCRIPTIONS OF PREFERRED EMBODIMENTS

[0035] Embodiments of the present invention will now be described with
respect to the Figures which depict what is now considered the best mode
to make and use the invention. Those skilled in the art will recognize
that the embodiments described and depicted are capable of being modified
and altered such that the present invention should not be limited to the
precise details.

[0036] One embodiment of the present invention, directed to a device for
performing a chromatographic separation and a nuclear magnetic resonance
analysis on a sample, generally designated by the numeral 11, is depicted
in FIG. 1. The device 11 comprises a closed chromatographic assembly 13,
conduit means 15, Nuclear magnetic resonance assembly 17 and control
means (in the form of system controller) 19.

[0037] Turning first to the chromatographic assembly 13, the
chromatographic assembly is closed in the sense that the chromatography
is performed in a vessel of pipe closed and contained from the
atmosphere. The chromatographic assembly 13 has an input in the form of a
needle 21, an outlet 23, one of more pumps 37 and a chromatographic
device 27.

[0038] The input 21 is for receiving one or more samples. The output 23 is
for discharging one or more separated samples. The chromatographic device
25 is a column, cartridge, capillary or other inline plumbed separation
device. Such separation devices are typically packed with particles,
beads, porous monolith and the like, although capillary type devices may
rely on internal wall structures. The chromatographic device 25 is for
separating the sample to form one or more separated samples having
retention time data.

[0039] Conduit means 15 is in fluid communication with the outlet 23 of
the closed chromatographic assembly 13 for conveying the one or more
separated samples to the nuclear magnetic resonance assembly 17. Conduit
means 15 encompasses tubing, piping, conduits, capillaries and all
associated fittings, valves, and ancillary supporting components and the
like for placing components and assemblies in fluid communication. As
used herein, the term "fluid communication" means plumbed together, as in
linked by pipes, tubing and the like, to move fluids there between.

[0040] As depicted, conduit means 15 has trapping means 31 for holding a
separated sample to form a held separated sample and placing said held
separated sample in said nuclear magnetic resonance assembly 17. The
trapping means 31 forms a held separated sample and a passed separated
sample. The passed separated sample is discharged from the device at
trapping means waste 33. Preferred trapping means comprise a trapping
column 35 or a separated sample loop [not shown] or a vessel [not shown].
The trapping column 35 is plumbed to a trapping means 31 allowing
unwanted fluids to be discharged and desired deuterated reagents to elute
analytes.

[0041] A preferred trapping means 31 is in fluid communication with
nuclear magnetic resonance reagents, deuterated reagents, by means of
pumps represented by numeral 37. The trapping means 35 releases the held
separated sample in the nuclear magnetic resonance reagents to form one
or more deuterated separated samples for nuclear magnetic resonance
analysis.

[0042] Nuclear magnetic resonance assembly 17 receives one or more
separated samples defined by retention times and producing nuclear
magnetic resonance data for the one or more separation samples. The
nuclear magnetic resonance assembly comprises a NMR probe 41 and a NMR
console 43. The NMR console is a control unit for the NMR assembly 17.
The NMR probe 41 is a subsystem with at least one end located within a
very strong and highly homogeneous primary magnetic field (B_zero field),
where the probe is tuned to detect the magnetic resonance of protons, or
potentially other atomic species, on the analyte molecule. A "flow probe"
such as the MRM microcoil probe contemplated for use here, incorporates a
very low-volume (few-microliter) flow cell having an inlet port and an
outlet port. The inlet port is in fluid communication with the output of
conduit means 15. The outlet port may simply lead to a waste-collection
device, or may lead to a vial or well-plate which may be used to recover
sample for other purposes, as NMR detection is a substantially
non-destructive technique. A flow probe eliminates the mechanical
manipulations associated with transporting "NMR tubes" into and out of
the interrogation region of the NMR spectrometer. Tube-based NMR
measurements are part of the original or classic mode of use, but to
achieve online connectivity and concommittant throughput enhancements, a
flow probe is a practical necessity. It allows the probe hardware to be
installed once (and in many cases, to be shimmed just once), and not
physically moved to perform subsequent analyses. Rather, only the
solvated sample is conveyed into and out of the interrogation region of
the probe, using liquid flows derived from pumps. Such flows may be
redirected, or stopped-and-started, using chromatography-grade switching
valves.

[0043] Control means (also referred herein as system controller) 19 is in
signal communication with the closed chromatographic assembly 13, conduit
means 15 and the nuclear magnetic resonance assembly 17. The Control
means 19 receives retention time data and nuclear magnetic resonance data
and associates the retention time data and nuclear magnetic resonance
data to at least one of the sample and the separated sample. The control
means 19 is a computer or CPU and supporting software, firmware and
instructions. The computer or CPU may be a personal computer, mainframe,
server or integral with one or more assemblies of the device 11.

[0044] The device 11 further comprises at least one second detector in the
form of mass spectrometer 47 and photodiode array detector 49. Each
detector, mass spectrometer 47 and photodiode array detector 49, is in
fluid communication with the conduit means 15 and in signal communication
with the control means 19. The second detector produces second detector
data, and the control means associates the second detector data with the
retention time data and nuclear magnetic resonance data to at least one
of said sample and said separated sample.

[0045] A preferred device 11 uses the photodiode array detector 49 as a
peak detector. The photodiode array detector 49 is in fluid communication
with the conduit means 15 and is in signal communication with the control
means 19. Conduit means 15 has a fraction selection or "slicing" valve 51
in signal communication with control means 19 which works in conjunction
with the control means 19 and the photodiode array detector to isolate
peaks. The fraction selection valve 51 has one or more sample holding
loops 53 to park or temporarily hold a peak of interest before
discharging such peak to the trapping means 31.

[0046] The photodiode array detector 49 produces one or more signals
corresponding with an analyte of interest or a potential analyte of
interest in a separated sample to isolate the separated sample to form an
isolated separated sample. The photodiode array detector 49 signal
prompts the control means 19 to direct the isolated separated sample to
at least one of the nuclear magnetic resonance assembly 17 and mass
detector 47. Preferably, fraction selection valve 51 receives the
isolated separation sample and forms isolated separated sample aliquots,
and directs at least one isolated separated sample aliquots to the
nuclear magnetic resonance assembly 17 and the mass detector 47 such that
said isolated separated sample aliquot is associated by control means
with nuclear magnetic resonance data, the photodiode array detector data,
the mass spectrometer data and retention time data.

[0047] With reference to FIG. 1, a hierarchical system-control arrangement
is shown, wherein a set of functional modules is implemented, each with
its own embedded, real-time controller (typically a microcontroller or
microprocessor executing programmed instructions according to, and
embodied within, embedded firmware). This set of functional modules is,
in turn, responsive to a host or supervisory controller, most typically
implemented as a computer workstation, which resides in electrical
continuity (including wired and/or wireless communication) with the
respective modules. The supervisory controller implements, via programmed
software, the user-interface for the human operator, thus allowing an
analyst to specify how a chromatography separation is to be accomplished,
and how the resulting data streams and liquid fractions are to be treated
and coordinated.

[0048] Importantly, this controlling interface includes, in a preferred
embodiment, the assimilation of data streams from both a photodiode array
UV-Visible absorbance detector ("PDA"), and a mass spectrometer of a
selected architecture, which may, for convenience, be a benchtop
single-stage quadrupole mass analyzer ("mass detector" or "MS"). These
two analyte-detection subsystems, responsive to the supervisory
controller, provide the controller with a window upon the liquid-phase
separation, which is used to steer the actions taken by the
fraction-selection valve and by the other modules within the system. The
integration of control at the supervisory level is critical to achieving
closely-coordinated action throughout the system, and will be seen as
substantially fundamental to the execution of a full in-line
structure-elucidation.

[0049] It will be recognized that, at the option of the user, the detector
types indicated above may be replaced or augmented with other detector
types as appropriate to the end-use application intended by the user.
Examples might be the substitution of an evaporative light-scattering
detector ("ELSD") or charged-aerosol detector ("CAD") to better address
analyte detection in situations where substantially no UV-chromophore is
present, or the substitution of a tandem mass spectrometer ("MS-MS") in
place of, or augmenting, the single-stage mass spectrometer described
above. The tandem mass spectrometer may comprise one or more of any of
the known mass spectrometer architectures (such as quadrupole mass
analyzers, time-of-flight mass analyzers, sector analyzers, ion-cyclotron
resonance analyzers, or others), and may include one or more collision
chambers to augment analyte fragmention between mass analyzer stages, as
is known in the art. The mass spectrometer may further implement one or
more ion-mobility spectrometry ("IMS") drift-tubes between mass-analyzer
stages, to achieve further dimensions of resolution of analytes. Such
mass spectrometers are manufactured by Waters Corporation, with
commercially-available examples being the Synapt G1 and G2 series of
instruments.

[0050] At the left side of FIG. 1 is a first chromatography system,
hereinafter referred to as the primary chromatography system, configured
so as to carry out a "complete" chromatography separation, in that each
of the following requisite functions is present: (1) non-deuterated
solvent gradient generation and delivery, (2) sample management including
sample injection, (3) analyte separation on a column containing an
appropriate stationary phase, (4) analyte detection mediated by a first
and a second detection subsystem (UV-Visible absorbance detection and
mass spectrometric detection, respectively, in this exemplary
embodiment).

[0051] The analyst's sample is first maintained and then introduced by way
of the sample manager within the primary chromatography system, which
performs the injection of the commanded sample volume into the primary
chromatography system mobile-phase stream. Typically, in analytical
chromatography, the sample volume is configured such that the sample mass
injected is a small enough value to avoid overloading the capacity of the
column, while being large enough that the sample components can be
visualized with sufficient signal-to-noise at the detector(s). Depending
upon the scale at which the primary chromatography separation is
conducted, the analyte-of-interest may, or may not, be present in
sufficient mass to accomplish the intended NMR spectroscopy based upon a
single sample-injection event (see below for sample-accumulation
functionality).

[0052] Intervening between the PDA detector and the mass detector is a
four-port flow-splitter, such as that commercialized by Waters
Corporation. Whereas the PDA detector, which is a substantially
non-destructive analyte detector, experiences the through-flow of the
entirety of the mobile-phase stream emerging from the separation column,
the mass detector (which is a destructive analyte detector) sees or
experiences only a minor proportion of that mobile-phase stream (a "split
ratio" of 15:1 is used in one exemplary embodiment, where only about 6%
of the analytical mobile-phase stream is directed to the mass detector.
The balance of the flow, or about 94% of the analytical stream, is
redirected to a different, selected outlet port.) Inherent in the design
of the Waters flow-splitter is a port for providing a makeup solvent
flow, such that the minor percentage of analytical flow which is tapped
from the analytical stream for mass spectral analysis is conveyed
efficiently to the mass detector, and that the analyte arrives at the
mass detector in a solvent environment which is optimized for the mode of
mass spectrometric analysis being employed.

[0053] In the illustrative embodiment of FIG. 1, this makeup flow is
sourced from a 515 pump module, which is compact and comparatively
inexpensive. A representative flow rate sourced by this pump might be
0.20 mL per minute, although that value is not intended to be unique or
limiting within this disclosure. The bulk of the mobile-phase stream
emerging from the analytical column and transiting the flow-splitter is
emitted from the splitter through a port which is in fluid communication
with a rotary shear-seal selection valve, also known in chromatography as
a "switching valve". This valve is indicated on FIG. 1 as AMAV1
("automated motorized auxiliary valve #1"). When the detector(s) indicate
to the system controller that the analyte-of-interest is not present in
the mobile-phase stream, this selection valve is oriented such that the
mobile-phase stream is directed to waste, without transiting the
accumulator-loop of AMAV1. Only when the detector(s) indicate(s) the
presence of the analyte-of-interest, is AMAV1 transiently switched so as
to capture the mobile-phase stream into the accumulator-loop volume,
thereby arresting the analyte-of-interest within that loop. AMAV1 is also
referred to hereinbelow as a "fraction-slicing valve", as a result of
this functionality.

[0054] With the sample-focusing capability resident in the downstream
trapping arrangement, the AMAV1 loop volume may be selected to be
multiple times the expected volume of an eluting chromatography peak,
such that the analyte-of-interest resulting from multiple, serial
chromatography separations can be accumulated there, prior to re-focusing
and entrance into the NMR spectrometer. This accumulation capability,
followed by sample re-focusing is, to our knowledge, a unique attribute
of the instant invention. This accumulation capability speaks to matching
the mass-loading of the chromatography separation with the analyte mass
requirements of the NMR spectrometer. The volume, geometry,
particle-size, and other properties of the packed-bed used for analyte
trapping may be quite different from those selected for the primary
chromatography separation column. Those attributes of the trapping column
will typically be selected to achieve a loading capacity consistent with
the analyte-mass needs of the NMR spectrometer. See also the section
discussing the concept of impedance-matching between LC separation and
NMR analysis. The primary analytical column may be operated at sample
loads which are less than that required by the NMR spectrometer, and that
multiple instances of primary chromatography separation may be undertaken
to accumulate the necessary mass of separated (fractionated) analyte
within the accumulator-loop at AMAV1.

[0055] In one preferred embodiment, the scale of the primary
chromatography separation is chosen such that a single instance of
elution of the analyte-of-interest is captured within the
accumulator-loop at AMAV1. In this case, "accumulation" corresponds to
the isolation of substantially one chromatographic "peak" or band within
that loop. Generically, during the analyte-accumulation phase of system
operation, the pump module labeled "515 Loop-Pump" is maintained at a
flow rate of zero. Once the analyte-accumulation phase is complete, the
accumulator-loop of AMAV1 will have been charged with analyte
corresponding to the results of one or more primary chromatography
separations. Expulsion of the loop contents is accomplished by switching
AMAV1 to the state which places the accumulator loop on-line with the 515
Loop-Pump, and by providing a loop-expulsion flow rate at the 515
Loop-Pump. Consistent with the at-column dilution principles recited in
Wheat, T. et. al. in U.S. Pat. No. 6,790,361 (incorporated herein by
reference), an aqueous flow derived from the "515 Dilution Pump" is
summed into the loop-expulsion flow at the "At-column Dilution Tee"
upstream of the trap column. Provision of this diluent reduces the
solvating strength of the mobile phase and allows the analyte-of-interest
to be chromatographically adsorbed or trapped on the reversed-phase
trapping column affixed to AMAV2 ("automated motorized auxiliary valve
#2"). Advantageously, the properties of this diluent flow are modified to
promote highly-efficient trapping of the analyte-of-interest. For
example, if the analyte resides as an acid in solution, the solution (the
diluent mobile phase) may be acidified, through the use of a solvent
modifier, to decrease the solubility of the analyte and promote the most
efficient trapping behavior. Once the analyte is bound, or trapped,
on-or-against the stationary phase, it is possible to eliminate the
solvent modifier and wash the trapped analyte with, for example, neat
D2O. The goal of this washing is to condition the retained analyte
into a regime most advantageous to support high-quality NMR spectroscopy.
In most cases, elimination of buffers or other solvent modifiers is an
important step toward achieving high-quality NMR results. For the instant
NMR application, the aqueous diluent flow may be further defined to
comprise a deuterated aqueous flow (ie. D2O). This aqueous flow is
provided over a timeframe such that the volume dispensed is sufficient to
convey the analyte toward and onto the trap column, and further to rinse
or flush the trap column to remove H2O and substitute it with
D2O.

[0056] Provision of an aqueous diluent at the at-column dilution tee is
consistent with a reversed-phase mode of trapping at the trap column per
Wheat et. al., although other modes of analyte trapping such as
normal-phase trapping are contemplated. The loop-expulsion and
analyte-trapping operation is allowed to proceed until the entire loop
volume at AMAV1 has been fully flushed. Accomplished under system
control, this process results in the analyte-of-interest being focused
substantially at the head of the trapping column. In the process of
trapping, the analyte may have been transported some measurable distance
from the chromatography system toward the NMR flow-probe entrance-port.
This transport step can be achieved with substantially no zone-broadening
impact, as the trapping process exerts a pronounced re-focusing effect
upon the analyte. To our knowledge, this is another unique attribute of
the instant invention.

[0057] During the trapping process, the liquid stream bearing the analyte
and the diluent transits the trap column and emerges from AMAV2 to waste.
Also in a first preferred embodiment, during the trapping process, the
deuterated-organic pump "B" belonging to the Deuterated Gradient Binary
Solvent Manager ("Deuterated BSM") is actuated to achieve a non-zero flow
rate, such that the fluid path intervening between AMAV2 and the outlet
of the NMR flow probe is pre-filled with the deuterated organic phase, in
preparation for analyte elution from the trap.

[0058] After the trapping phase is completed, the loop-pump and the
at-column dilution pump are respectively brought to the zero-flow state.
Typically, the deuterated-organic pump is also brought to a zero-flow
state at this time. AMAV2 is then switched to the position which places
the trapping column in fluid communication with the deuterated gradient
BSM and with the NMR flow-probe. Under system control, the deuterated
gradient BSM organic pump is then actuated to deliver deuterated organic
solvent in a volume as required to elute the analyte off the trap and to
transport the analyte to the NMR detection cell. Once that volume has
been delivered, the pump is arrested, causing the analyte peak to be
"parked" in the NMR cell.

[0059] It is an option to incorporate an additional valve at the outlet of
the NMR flow probe, such that "coasting" or "skidding" of the analyte
peak during the parking process is substantially avoided. Skidding can
arise from the relaxation or decompression of previously-compressed
volumes of solvent (compressed during delivery of liquid through the trap
and associated tubing). The use of a controllable valve to block flow at
the exit of the NMR flow-probe can be advantageous to arrest this
skidding response, thereby improving the positioning accuracy of the
"park". Also, where the term "deuterated organic solvent" appears above,
that solvent may indeed be neat deuterated organic solvent, such as
DMSO-d6 or CD3CN, or it may be an organic-organic or an
organic-aqueous deuterated mixture which has sufficient solvating
strength to elute the analyte-of-interest from the trap column in a
substantially-sharp zone or band. The use of a gradient BSM at this
location in the system facilitates selection of a desired solvating
composition, under program control. In many cases, the neat
deuterated-organic may be most desirable for this purpose, but options
and alternatives exist, and may be programmed into use by the analyst.

[0060] Once the analyte-of-interest is parked within the interrogated
region of the NMR flow-cell, the NMR controller can undertake automated
shimming, followed by spectral acquisition and post-processing. Known NMR
controllers have the ability to accumulate spectra for the period of time
necessary to achieve a desired S/N ratio, and to perform a variety of
post-processing steps. Some or all of the post-processing which is
accomplished after spectral acquisition may occur within the host
workstation.

[0061] Preferably, the host workstation, which is the supervisory
controller for all of the foregoing operations, has a scope of analysis
which is broad enough to assimilate at least UV spectral data, mass
spectral data, and NMR spectral data (and potentially other data streams
as well), and to coordinate the reporting of that data to a software
functionality that may provide structure-elucidation along with a
confidence-level, as the output of the analysis. Unlike prior-art
approaches to the preparation and handling of samples for NMR
spectroscopy, which may involve many manual transfers and manual or
semi-automated processing steps, and may occupy multiple systems in
different locations, an integrated and fully-automated system can provide
a traceable trail which connects a relatively "raw" incoming sample
mixture with a finished output such as an elucidated structure or
absolute compound identification.

[0062] Changes to the system configuration depicted in FIG. 1 may be
contemplated while remaining within the spirit and scope of the instant
invention. One such change is depicted in FIG. 2. In the system of FIG.
2, the module indicated as "515 Dilution Pump" has been removed, and its
function replaced by pump "A" of the deuterated Gradient BSM #2. This
change eliminates one hardware module, thus making the system slightly
less complex, or slightly less expensive to implement. The change,
however, implies that pump "B" of the deuterated Gradient BSM will be
used exclusively to elute the analyte from the trap column (i.e. blended
solvent mixtures produced by the coordinated actions of pumps "A" and "B"
of the deuterated Gradient BSM will not be available by program to elute
sample from the trapping column.) For many applications, a neat organic
solvent delivered by pump "B" may be quite acceptable for this elution
task, or a pre-blended solution which is delivered solely by pump "B" may
be used.

[0063] In yet another preferred embodiment--In an era of "fast
chromatography", particularly chromatography which is fast relative to
the anticipated NMR acquisition time, it is of significant interest to
accumulate analyte from multiple (typically sequential) primary
chromatography separations, derived from respective instances of sample
injection, and to efficiently co-add the separated (fractionated) analyte
material to produce the purified, concentrated sample mass required for
NMR analysis. In this way, the sample-mass and/or sample-volume
limitations of the primary chromatography separation can be respected, so
as to deliver a usefully high-quality separation which preserves the
available chromatographic resolution. Maintaining chromatographic
resolution is an important contributor to maximizing the purity of the
collected fraction, and thereby minimizing background signal at the NMR
unrelated to the analyte-of-interest. It has been stated in the foregoing
description that the analytical system of FIG. 1 or of FIG. 2 can be
configured and operated in a mode where the accumulator-loop at AMAV1 is
sized to accumulate analyte from multiple, sequential,
primary-chromatography separations. It will be recognized that an
accumulator-loop has a finite volume, which determines an upper bound on
the number of primary chromatography separations from which analyte can
be extracted or fractionated. In the case of relatively dilute analyte
solutions, increased analyte mass may still be desired, even when the
accumulator-loop is filled (from a volume standpoint). Overfilling the
accumulator-loop results in direct analyte loss to waste, which is
undesirable. In a preferred embodiment, the system of FIG. 1 or of FIG. 2
can be operated such that when the accumulator-loop at AMAV1 is filled
(from a volume standpoint), the system controller undertakes a trapping
operation which focuses and retains the analyte, while directing the
solvent volume to waste. It will be recognized that while the trapping
column has a finite trapping capacity which relates to the analyte mass
applied, the trap can process or throughput an almost arbitrarily large
volume of liquid solvent, thereby substantially overcoming the volume
limitation of the accumulator-loop at AMAV1. Once analyte trapping is
accomplished, the accumulator-loop at AMAV1 is effectively restored to
its "empty" state, and is ready to receive new aliquots of analyte from
the primary chromatography system. Particularly in the case of
environmental analysis, where the analyte-of-interest may reside at a low
level within the environmental primary-chromatography sample, multiple
cycles of "accumulate-and-trap" may be carried out, such that the
trapping column is taken close to its saturation capacity of analyte.
Again, the trap column geometry and stationary phase are selected to
optimally match the analyte mass requirements of the NMR spectrometer.
This approach may achieve a substantial degree of decoupling between the
scale of the primary chromatography separation and the analyte
requirements of the NMR spectrometer. This substantial decoupling may
itself overcome one of the perceived limitations to accomplishing the
hyphenation of chromatography with NMR spectroscopy. It will be readily
apparent that within the spirit and scope of the instant invention, it is
an option to perform either: (a) multiple analyte accumulations at the
AMAV1 loop, followed by a trap event, or (b) single analyte accumulations
at the AMAV1 loop, each followed by a respective trap event, or (c) any
combination of (a) and (b), as specified by the user. It should also be
noted that there are chromatography conditions which must be met in order
to achieve highly-efficient sample aggregation at the trap (i.e.
minimizing sample loss or breakthrough at the trap), in concert with
efficient sample focusing. In a recent scientific publication (Sandvoss
et. al., Magn. Reson. Chem. 2005; 43: 762-770) the authors recited an
approach where a three-fold excess of water (unmodified water) was added
to a chromatographic eluent stream in order to attempt to trap analyte on
HySphere Resin GP (general-purpose polydivinylbenzene-based resin)
cartridges. These authors documented at length problems which they
encountered in obtaining sample aggregation in multiple-trapping
instances. Also, they pointed out that "as the polarity of the compounds
increases, the efficiency of the multiple trapping decreases." As the
eight authors of this paper practice within the sphere of pharmaceutical
industry analysis, one might accept these findings as indicative of the
current "state of the art". It will be noted that in the instant
invention, highly-efficient sample aggregation and a substantial absence
of trap breakthrough has been confirmed using appropriate in-line
detectors, and those behaviors arise as a consequence of careful
attention being paid to the following details. First, a very highly
retentive stationary phase (Oasis® HLB, Waters Corporation, Milford,
Mass., USA) is incorporated into the trapping column. Second, appropriate
selection of mobile phase modifiers for the diluent stream feeding the
at-column dilution tee is made, in order to optimize trapping behavior
beyond what would be achieved through the use of neat H2O or
D2O. Third, the ratio of diluent flow rate to analyte flow rate must
be extreme enough to ensure that trapping is accomplished (i.e. local
solvating strength is reduced sufficiently), independent of the solvent
environment from which the analyte was initially sliced. One should
recognize that when analyte is sliced from its elution position within
the primary chromatogram, a sampling of the instantaneous mobile phase
condition (in which that elution occurred) is likewise captured into the
slice-valve loop. That mobile phase condition accompanies the analyte
along the path toward the at-column dilution tee. The diluent addition,
and the at-column dilution tee geometry, must both be selected to achieve
the necessary reduction in solvent strength to allow the analyte to be
refocussed and trapped on the trap bed, independent of the location
within the primary chromatogram (i.e. the local solvent environment) from
which the analyte was sliced. A failure to produce sample aggregation (as
documented in the Sandvoss et. al. paper) is a measure of the failure to
achieve a chromatographic trapping condition at the trap column. When
trapping conditions are satisfied, the trap works properly, and analyte
aggregation and refocusing result.

[0064] Preferably, all of the capacity of the stationary phase within the
trapping column is available for retention of the substantially-purified
analyte-of-interest, in contrast to the situation at the primary
chromatography column, where a substantially cruder sample mixture is
applied, and where the analyte-of-interest may represent only a minor
component of the totality of the material applied during
primary-chromatography sample-injection.

[0065] With regard to FIG. 3, a system is shown which extends or augments
the system of FIG. 2 with an additional module comprising rotary
shear-seal valves and a plurality of external loops for respective
analyte accumulations. It will be recognized that the system of FIG. 2,
which implements a single analyte-accumulator loop at AMAV1, will
typically be limited to accumulating only a single analyte species, from
a single peak-of-interest, from a single or from multiple primary
chromatography separations. If multiple primary chromatography
separations are performed sequentially, typically the same analyte
species will be accumulated from the same peak-of-interest within each of
"N" respective primary separations. However, analytical scenarios are
readily anticipated where multiple peaks-of-interest may be present
within a primary chromatography separation, and where the co-addition of
different analyte types within a single accumulator loop is highly
undesirable.

[0066] In such scenarios, an augmented analyte-redirection and
analyte-accumulation capability as implemented in FIG. 3 may be usefully
employed. The functionality depicted within the added module corresponds
to that of a "1-of-N Demultiplexer/Multiplexer"arrangement, which inserts
fluidically into the system of FIG. 3 at AMAV1 ports 1 and 4,
respectively, thereby replacing the single loop which would otherwise be
shown connecting that pair of locations. The rotary shear-seal valves of
the Demultiplexer/Multiplexer are constructed so as to allow a single
fluid port, positioned at the center of the valve stator, to be
selectably placed in fluid communication with any one of "N" ports
radially disposed about the center of the stator. At the fluid-entrance
side of the arrangement (the Demultiplexer), this construction allows an
incoming fluid stream to be redirected to any one of "N" separate
analyte-accumulator loops. At the fluid-exit side of the arrangement (the
Multiplexer), this construction allows a selectable one-of-N
analyte-accumulator loops to exhaust fluid toward a single exit port. The
terms multiplexer and demultiplexer are drawn from the corresponding
functionalities which are known from digital electronics. Overall, the
coordinated action of the demultiplexing and multiplexing valves allows a
selectable one-of-N analyte-accumulator loops to be placed on-line for
analyte accumulation, at the request of the supervisory controller. As
that supervisory controller is assimilating data streams from the several
detector types, it can intelligently determine which of the "N" available
accumulator loops is selected to accumulate a particular peak within the
primary chromatography separation. In this way, multiple peaks can be
isolated and accumulated into respective accumulation loops, from a
single primary chromatography separation, or from a series of primary
chromatography separations. These respective analyte accumulations will
remain separate and distinct, and may be analyzed sequentially by the NMR
spectrometer, in successive, distinct cycles of loop-expulsion and
analyte trapping.

[0067] With reference to FIG. 4, an alternate embodiment of the primary
chromatography system is shown. The functionality depicted in FIG. 4 may
be inserted, in a substantially modular manner, into the systems of any
of FIG. 1 through FIG. 3, inclusive. FIG. 4 depicts a primary
chromatography system which is configured to enable the focusing (and
thus concentration) of dilute incoming samples, as might be required to
perform certain environmental analyses. In an analytical scenario where
the analyte-of-interest is a trace component within, for example, a water
sample, it may be necessary to process a relatively large volume of that
water sample in order to acquire enough analyte to carry out the intended
analysis. In the primary chromatography system of FIG. 4, a primary
trapping column is shown, the purpose of which is to achieve a first
coarse step of concentration of analyte. Other materials present within
the incoming sample may also undergo concentration at the primary trap,
and will be separated from the analyte-of-interest in subsequent stages
of chromatography. The primary trapping column incorporates a single
fluid flow direction for both loading (trapping) and for elution, in the
embodiment pictured. The terminology of "forward trapping, forward
elution" is often associated with this configuration. A secondary
focusing trap column is shown, which incorporates a preferred
bi-directional fluid flow configuration, where elution is accomplished in
the reverse direction from trapping ("forward trapping, back elution"),
through the use of a switching valve. It will be recognized that elution
of sample from the primary trap requires a transient increase in the
solvating strength of the eluent delivered by BSM #1. The module depicted
at BSM #1 may, alternatively, be a quaternary solvent manager ("QSM"),
thereby providing more alternatives to increasing the solvating strength
of the eluent stream delivered to the primary trapping column.
Correspondingly, the functionality indicated at BSM #2 could be
supplanted by a QSM. The at-column dilution tee and 515 pump module shown
are responsible for reducing the solvating strength of the eluent stream
emerging from the primary trapping column, in order that the material
released from the primary trap may be refocused on the secondary focusing
trap. At either of the trapping stages, sample material which is not
trapped, along with the incoming solvent, is directed to waste. The
back-elution which occurs at the second focusing trap may contribute to
better chromatographic resolution in the primary chromatography
separation, which is brought about by BSM #2 acting in concert with the
analytical column. In a system corresponding to that of FIG. 4, the
primary trapping column may be a relatively low-pressure device such as
an Oasis® cartridge, as the primary sample trapping occurs separately
from the primary chromatography separation (i.e. it is a separable
process antecedent to primary chromatography). In contrast, the secondary
focusing trap must be constructed to withstand the full operating
conditions extant during primary chromatography, as this trap will
participate directly in that process. This system configuration enables
efficient and automated processing of relatively-dilute (and
correspondingly, relatively large liquid-volume) incoming samples as
might be encountered in the environmental analysis realm.

[0068] With reference to FIGS. 5a and 5b, valve components and fluidic
interconnections are shown, which comprise a preferred embodiment for the
implementation of conduit means 15 as introduced in the context of the
system of FIG. 1. This preferred embodiment is optimized to achieve the
"impedance matching" behavior discussed above. In FIG. 5A, a trap valve
corresponding to AMAV2 of FIG. 1 is depicted along with a trapping column
or cartridge. The trap valve shown is of a six-port, rotary-shear-seal
design as is known in the art. The rotor in this valve incorporates 3
distinct etched pathways, and commutates between two distinct states, as
illustrated by the three solid and three dashed lines in the figure. Two
supplementary valves are also depicted in FIG. 5A, these being referred
to respectively as a "gas/liquid valve", and a "gas/D2O valve".
While these valves are also of a rotary-shear-seal design, and
incorporate 6 ports each, they differ from the trap valve in one
important respect. These latter two valves are constructed with rotors
which incorporate only two etched pathways, and which commutate between
two distinct states. This construction is illustrated by the two solid
lines and two dashed lines, on the respective figures. This valve
construction is known in the art as a "dual diverter-type valve". Also
shown in FIG. 5A is an at-column dilution tee, which allows at least two
input flow streams to be summed at the entrance side of the tee, and
optimally includes a mixing functionality such as a porous bed packed
with glass or ceramic beads or spheres. The function of this at-column
dilution tee is to blend a diluent stream with a chromatographic stream
in order to achieve a significant reduction in the solvating strength of
the chromatographic stream. The purpose of this reduction in solvent
strength is to achieve retention of an analyte on a chromatographic bed,
typically after that analyte has been released from a chromatographic bed
located upstream from the at-column dilution tee.

[0069] With reference to FIG. 5B, a fraction-selection valve is shown. In
this illustrative embodiment, the fraction-selection valve is implemented
as a 10-port, rotary-shear-seal type, as is known in the art. Also shown
in FIG. 5B is a flow splitter and its respective connections to other
modules in the system.

[0070] The sequence of operation, and the significance, of the components
illustrated collectively in FIGS. 5a and 5b is addressed immediately
below. The description of the sequence of valve operation is supported
and further clarified by reference to the tabulated valve-state
transition data appearing in Table 1.

[0071] The fraction-selection valve has a normal rotor position or valve
state of "1", as illustrated by the solid interconnect lines. In this
state, a chromatographic stream emerging from the flow splitter simply
transits the valve, entering at port 10 and exiting at port 1
respectively. No fraction-collection or slicing of analyte from the
chromatographic stream is accomplished with the valve in this state. When
this valve is commanded into state "2", illustrated by the dashed
interconnect lines, the flow-path through the valve changes such that the
chromatographic stream now transits the temporary storage or "slicing"
loop. That is, the flow which enters the valve at port 10 emerges at port
9, enters the loop and transits the loop, re-entering the valve at port
2, and exiting the valve at port 1. When the fraction-selection valve is
actuated only transiently into state "2", as guided by the in-system
detectors, the effect is to capture or store a narrow region of the
chromatogram into the temporary storage loop. The nomenclature of
"slicing" is used because a narrow region of the primary chromatogram has
been "sliced" out, so as to be available for processing elsewhere within
the conduit means. The contents of the temporary storage loop remain
in-place within the loop if the loop-expulsion pump connected to port 3
of the fraction-selection valve is held or maintained at a flow rate of
zero. When the system controller dictates that analyte trapping is to
commence, the loop-expulsion pump is provided with a non-zero flow rate
while the fraction-slicing valve is maintained in state "1", causing the
sliced fraction to be migrated out of the temporary storage loop, exiting
the valve at port 8 and proceeding to the at-column dilution tee of FIG.
5a. Diluent flow, typically in meaningful excess over the loop-expulsion
flow, is enabled to the diluent input of the at-column dilution tee.
Referring to FIG. 5A, with the gas/liquid valve maintained in state "2",
and with the trap valve maintained in state "TRAP", the diluted stream
bearing the analyte-of-interest is conveyed to the trap cartridge, where
the analyte is substantially retained. Again, the diluent flow which is
provided in excess can be configured with modifiers selected so as to
improve or maximize the trapping behavior at the trap column. When the
analyte-trapping phase has run to completion, typically under
time-programmed control, the loop-expulsion flow and the diluent flow are
typically reset to zero. The phase of operation referred to herein as
sample "polishing" will now be discussed. Analyte currently resident in
the trap column exists in a protonated solvent environment, which may
include modifiers such as buffers, salts, and the like. With the
gas/liquid valve transitioned to state "1", the gas/D2O valve may be
employed to allow replacement of the modified, protonated solvent
environment with a deuterated environment substantially free of
modifiers. That sequence is detailed immediately below. With the
gas/D2O valve in state "1", a flow of dry, inert gas is enabled to
the trap column, substantially expelling the protonated solvent from the
void-volume of the trap bed, to waste. Under time-programmed control, it
is at the option of the operator to simply expel liquid from the void
volume, or, with a more prolonged gas flow, begin to take the bed toward
a state of "dryness". In a preferred embodiment, the trap bed is not
taken to dryness, but is simply purged of any bulk amount of protonated
solvent (i.e. protonated solvent is generally expelled from the void
volume of the bed). Once this expulsion is accomplished, the gas/D2O
valve is transitioned to state "2", allowing a flow of deuterium oxide to
traverse the trap bed to waste. In a preferred embodiment, this deuterium
oxide flow is provided by a syringe-based infusion pump such those
produced commercially by Harvard Apparatus Inc. (Holliston, Mass., USA)
under the model designation PHD. It will be noted that unlike
larger-volume, continuous-flow pumps such as chromatography pumps, which
are typically constructed with solvent reservoirs and lengthy inlet
tubing lines which may be permeable to atmospheric contaminant species
such as water molecules, a glass-barreled syringe pump allows for fresh
deuterated solvent to be taken up directly from a previously-sealed glass
ampoule as shipped by the supplier, and quickly encapsulated with the
glass-syringe environment. This absolutely minimizes exposure of the
deuterium oxide or other deuterated species to water proton
contamination, improving the quality of the analyte deuteration process.
The volume of the glass ampoules in which deuterated solvents may be
ordered can be chosen so as to match the syringe barrel volume on the
syringe pump, so that a single-use/single-filling is achieved, and there
is no need to reseal ampoules after opening. This one-time-use procedure
maintains a very high quality of the deuterated phase, and is preferable
for routinely achieving high-quality NMR spectroscopy. Once a glass
ampoule has been cracked open, typically only a few seconds elapse before
the deuterated solvent contents are safely encapsulated within the glass
syringe barrel of a Harvard-type syringe pump. The piston seals on such a
pump are typically Teflon, but the seal is labyrinthine enough that in
practice, there is negligible gas transfer between the external
environment and the interior of the syringe. Because the trap bed volume
is so small, the flow rates and solvent volumes used to deuterate that
environment are correspondingly small, and thus a syringe volume can last
for at least a full day of operation, which is convenient for the user.
By perfusing the trap bed to waste with deuterium oxide (D2O), salts
or other modifiers are solubilized away, and likewise water protons which
may reside in the pore volume of the bed are equilibrated away. It is at
the option of the operator, under programmatic control, to incorporate as
few or as many cycles of gas purging, followed by D2O purging, as
are necessary or consistent with the quality of the desired NMR
spectroscopy. It is our experience that retained analyte can be
"polished" in this way to a quality which is only limited by the quality
of the incoming deuterated phases. Typically such phases are purchased to
a specification which includes the residual proton contamination present.
The lowest levels of proton contamination are typically associated with a
higher-quality, and somewhat higher-cost, reagent. The quality of that
reagent thus can be more-or-less directly translated into a reduction of
proton background in the resulting NMR spectroscopy. The use of an
analyte "polishing" sequence, to remove salts or other mobile phase
modifiers, typically also has a direct and measurable result on the NMR
spectral quality with respect to freedom from artifacts.

[0072] When such polishing is completed, it is at the option of the
operator, under programmatic control, to cause the trapped analyte to be
eluted to the NMR microcoil flow probe. It will be noted from FIG. 5A
that with the trap valve maintained in the TRAP state, flow of the
deuterated organic solvent DMSO-d6 is enabled from port 4 of the trap
valve to port 5, and thus enabled to purge the microcoil NMR probe which
is in fluid communication with port 5 of the trap valve. This flow of
DMSO-d6 is typically sourced by a Harvard Instruments glass-syringe
perfusion pump, for the same reasons recited above for the D2O pump.
The DMSO-d6 flow is typically of even lower flow rate, and lower total
volume, than the above-referenced D2O flow, which provides a long
useable interval of operation between refills, even when modest
syringe-barrel sizes are employed. The bathing of the NMR probe with neat
DMSO-d6 is useful from several standpoints. It generally ensures that any
prior precipitates are removed from the flow conduits, and also prepares
and prefills the entrance and exit conduits around the NMR flowcell with
the same solvent as the analyte will be eluted in, to minimize magnetic
susceptibility mismatch which can lead to poor (broad) NMR spectral
linewidths. Prior to DMSO-d6 elution, the trap bed is given a final purge
with inert gas, thus allowing the incoming DMSO-d6 solvent front to
arrive in as sharp a configuration or "front" as possible, without mixing
or blending on the leading edge with a pre-existing D2O phase.
Following this inert-gas purge to waste, the trap valve is transitioned
to the BACK ELUTE state, and flow from the DMSO-d6 syringe pump is
enabled. This flow will be seen to back-elute the trap (i.e. in the
reverse direction from the trapping direction) into port 6 of the trap
valve, and out via port 5 to the NMR microcoil probe. Back-elution is
useful for at least the following reason. When trapping analyte with a
very highly-retentive phase, under proper trapping conditions, analyte
will be retained at, or very close to, the extreme inlet end of the trap
column. When that region of the trap column bed becomes saturated with
analyte, new incoming analyte will "spill over" into a subsequent region
of the column bed, penetrating slightly further into the bed than the
first portion of the analyte. As the trap bed slowly fills with retained
analyte, this spillover will continue to occur, with successive sections
of the bed becoming populated with analyte. In order to produce the
narrowest and most intense analyte elution band, irrespective of the mass
of analyte which is present, it is an advantage to back-elute the bed
with a very strongly-solvating mobile phase. The back-elution process
will scavenge the spillover analyte as the strong solvent migrates
through the bed, and will refocus the elution in a highly beneficial way.
The swept-up spillover will exit the column along with the preponderance
of analyte trapped at the column head, thus maximizing the amount of
analyte per unit volume of eluting solvent. Given proper selection of
trap-column bed-volume, this narrow, intense band back-eluted from the
trap column may have a volume at half-height of only some 6 microliters.
In contrast, that trapped analyte may have resided in the primary
chromatography separation in a band of some 100 microliter volume,
measured at half-height. This significant reduction in the volume over
which the sample mass is distributed, coupled with negligible (low- or
substantially no-) loss of sample mass from the primary separation, is a
statement of the "impedance matching" which has been accomplished between
the primary chromatography separation, and the microcoil NMR probe. The
volume scales and concentration scales of the two processes (primary
chromatography, and microcoil NMR detection) are very disparate, but are
bridged efficiently by an appropriate impedance-matching mechanism, as
described above. Appropriate impedance matching is fundamental to
achieving good utilization of sample mass, such that NMR spectra can be
acquired with good S/N, while avoiding mass-overloading of the primary
chromatography separation. It will be further noted, with respect to FIG.
5a, that an in-line capillary-scale UV absorbance detector can usefully
be incorporated into the flow path leading from trap valve port 5 to the
microcoil NMR probe. As shown below, such a detector can be used as an
in-line diagnostic tool to confirm the presence of analyte traversing the
path to the NMR probe, prior to NMR spectroscopy being performed. Such
capillary-scale UV absorbance detectors can be made small and relatively
inexpensively, and the diagnostic utility which they can provide can be
meaningful in practice.

[0073] With reference to FIG. 6A, a series of detector data streams is
depicted, to illustrate aspects of system operation. Shown is an example
of a successful mass-directed peak collection, followed by on-line
trapping and elution. FIG. 6A is an overview, and is followed by FIG. 6B
which expands a region-of-interest comprising minutes 4 through 9,
thereby allowing better viewing of details of certain signals.

[0074] With reference to FIG. 6A, the bottom or lower-most signal trace
originates from positive-electrospray mass spectrometry (noting again
that the mass spectrometer makes use of a small fraction of the total
primary chromatographic eluant stream which transits the chromatography
column and the PDA detector. That small fraction is apportioned by the
flow splitter shown in FIG. 5B.). In mass-directed fractionation, the
sensitive and highly-selective mass spectrometer detection guides the
operation of the fraction-selection valve. The next pane above the MS
signal is the signal from the PDA detector. The next pane above the PDA
signal is the signal from the evaporative light-scattering detector,
which, per FIG. 5B, is located along the chromatographic waste path
downstream of the fraction-selection valve, where it can detect the
presence or absence of materials in the chromatographic stream which is
being manipulated by the fraction-selection valve. The next pane above
the ELSD signal (the top-most pane) is the signal originating from the
in-line capillary-scale UV absorbance detector, representative of the
analyte band being manipulated toward the NMR microcoil probe. As the
elution flow rate from the trap is only 2 microliters per minute, the
peak width, expressed in volume units, represented by this signal trace
is only 6 microliters at half-height.

[0075] With reference to FIG. 6B, the set of signals of FIG. 6A is
expanded for better visibility, in the interval encompassing minutes 4
through 9 of the acquisition. In the ELSD signal (second from top pane),
one can clearly see the heart-cut of the peak where analyte has been
sliced from the primary chromatography stream. A small amount of the
leading and trailing edge of the primary peak remains, but the vast
majority of the signal (and thus the sample mass) has been sliced out of
the primary chromatography stream, and into the transient storage loop,
where it is not detected by the ELSD. The PDA detector signal (second
from bottom) shows a very strong peak in the primary chromatogram, which
is clipped or saturated at about 3 absorbance units, and therefore does
not exhibit the characteristic Gaussian peak shape of intensity. The
lowermost pane (positive electrospray MS), shows the detected primary
chromatography peak with an overlay of the slicing action which was
commanded (i.e. the "start" and "stop" window asserted to the
fraction-selection valve. The uppermost pane shows no meaningful signal,
as the time-window of minutes 4 through 9 of the analysis was an inactive
time from the standpoint of the in-line capillary UV detector.